This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The overall research goal in Rye's lab is to understand how GroEL, in conjunction with its partner protein GroES, utilizes the energy of ATP hydrolysis to promote protein folding of fully GroEL-dependent substrate proteins (so-called "stringent" substrates). Both GroEL and GroES are seven-fold, ring shaped oligomers. GroEL captures a folding intermediate on one of its open rings, then binds ATP and the co-chaperonin GroES, resulting in the encapsulation of the substrate protein within an enclosed GroEL-GroES complex. How sub-strate encapsulation by GroES is accomplished and precisely what GroEL does to a substrate protein that drives productive folding remain poorly understood. Rye has approached these problems using a combination of site-directed mutagenesis, chemical modification and fluorescence spectroscopy (Lin and Rye, 2004;Lin et al., 2008). The inability of previous studies to trap and study a pre-triggered state of the GroEL-GroES complex is not surprising, given that wild-type GroEL populates this allosteric state only transiently (for a hundred millisec or so), much too short a time for ready manipulation. By contrast, our modified GroEL variant appears to move through this transition at a considerably slower rate. We propose that a high-resolution look at this new GroEL variant, exploiting the technical advances the NCMI has made in examining molecules like GroEL by cryo-EM, is likely to provide an unprecedented snap-shot of a critical allosteric state of the GroEL-GroES machine about which we currently know very little.